cellular/molecular iqgap1regulatesnr2asignaling,spinedensity, … · 2011. 6. 8. ·...

Cellular/Molecular

IQGAP1 Regulates NR2A Signaling, Spine Density,and Cognitive Processes

Can Gao,1 Shanti F. Frausto,2 Anita L. Guedea,1 Natalie C. Tronson,1 Vladimir Jovasevic,1 Katie Leaderbrand,1Kevin A. Corcoran,1 Yomayra F. Guzmán,1 Geoffrey T. Swanson,2 and Jelena Radulovic11Department of Psychiatry and Behavioral Sciences, The Asher Center for Study and Treatment of Depressive Disorders, and 2Department of MolecularPharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

General or brain-region-specific decreases in spine number or morphology accompany major neuropsychiatric disorders. It is unclear,however, whether changes in spine density are specific for an individual mental process or disorder and, if so, which molecules confersuch specificity. Here we identify the scaffolding protein IQGAP1 as a key regulator of dendritic spine number with a specific role incognitive but not emotional or motivational processes. We show that IQGAP1 is an important component of NMDAR multiproteincomplexes and functionally interacts with the NR2A subunits and the extracellular signal-regulated kinase 1 (ERK1) and ERK2 signalingpathway. Mice lacking the IQGAP1 gene exhibited significantly lower levels of surface NR2A and impaired ERK activity compared to theirwild-type littermates. Accordingly, primary hippocampal cultures of IQGAP1�/� neurons exhibited reduced surface expression of NR2Aand disrupted ERK signaling in response to NR2A-dependent NMDAR stimulation. These molecular changes were accompanied byregion-specific reductions of dendritic spine density in key brain areas involved in cognition, emotion, and motivation. IQGAP1 knock-outs exhibited marked long-term memory deficits accompanied by impaired hippocampal long-term potentiation (LTP) in a weakcellular learning model; in contrast, LTP was unaffected when induced with stronger stimulation paradigms. Anxiety- and depression-like behavior remained intact. On the basis of these findings, we propose that a dysfunctional IQGAP1 gene contributes to the cognitivedeficits in brain disorders characterized by fewer dendritic spines.

IntroductionIn the CNS, specific patterns of molecular signals arise from lo-calized scaffolding of signaling modules to distinct membranereceptors and determine neuronal morphology and function.IQGAP1 serves as a scaffold for several signaling pathways, suchas Lis1, Cdc42, B-Raf, and extracellular signal-regulated kinase(ERK), which regulate spine density and show marked abnormal-ities in psychiatric illnesses (Kholmanskikh et al., 2006; Reiner etal., 2006; Ryu et al., 2008; Ide and Lewis, 2010; Yuan et al., 2010).We therefore hypothesized that IQGAP1 plays an important rolein dendritic spine regulation and behavior. IQGAP1 belongs tothe IQGAP family of molecular scaffolds named for their homol-ogy to GTPase-activating proteins (GAPs) and isoleucine/glu-

tamine (IQ) (calmodulin-binding) motifs (Briggs and Sacks,2003). Three isoforms have been described in mammals (Brownand Sacks, 2006; Brandt and Grosse, 2007; Wang et al., 2007),with IQGAP1 being identified first in 1994 (Weissbach et al.,1994). Mammalian IQGAP1 is a 190 kDa protein containing sev-eral domains that enable association with many binding partners(Brown and Sacks, 2006). These motifs include a calponin ho-mology domain, responsible for actin binding; a WW motif,which is necessary for the association with ERK2; a calmodulin-binding IQ domain; and a RasGAP-related domain that binds thesmall GTPases Cdc42 and Rac1 (Fukata et al., 1997; Owen et al.,2008). In addition, IQGAP1 binds to and regulates the functionsof E-cadherin, �-catenin, and cytoplasmic linker protein 170(Ren et al., 2005). Based on findings from multiple cell lines,IQGAP1 is considered to play pivotal roles in cell adhesion, migra-tion, and polarization (Noritake et al., 2005). IQGAP1 also regulatesmitogen activated protein kinase signaling (Roy et al., 2005) by di-rectly binding to B-Raf (Ren et al., 2007), mitogen-activated proteinkinase kinase (MEK) (Roy et al., 2005), and ERK (Roy et al., 2004).ERK is activated when Ca2� influx promotes direct binding ofIQGAP1 to the ERK activator B-Raf (Ren et al., 2008).

Despite its broadly recognized roles in various cell types, thefunction of IQGAP1 and associated signaling in the adult CNS isunknown. Yet, it has become increasingly recognized that the spe-cific cellular actions of signal transduction pathways critically de-pend on their compartmentalized regulation by scaffolding proteins.We showed recently that relocation of IQGAP1 from dendriticspines to the shafts parallels impairments of N-cadherin-mediated

Received March 14, 2011; revised April 4, 2011; accepted April 11, 2011.Author contributions: C.G., G.T.S., and J.R. designed research; C.G., S.F.F., A.L.G., N.C.T., V.J., and K.L. performed

research; C.G., S.F.F., A.L.G., N.C.T., V.J., K.A.C., Y.F.G., and G.T.S. analyzed data; C.G. and J.R. wrote the paper.This work was supported by the National Alliance for Research on Schizophrenia and Depression (C.G.), a Diver-

sifying Faculty in Illinois Award (S.F.F.), NINDS Grant NS071952 (G.T.S.), NIMH Grant MH073669 (J.R.), and Dunbarfunds (J.R.). We thank Drs. Andre Bernards (Massachusetts General Hospital Cancer Center, Boston, MA) and WadieBahou [State University of New York (SUNY) at Stony Brook, Stony Brook, NY] for providing the IQGAP1 knock-outs,Dr. Stefano Vicini (Georgetown University, Washington, DC) and Gulcan Akgul (SUNY Stony Brook) for providingNR2A-GFP, Dr. Zhong Xie and Dr. Peter Penzes for providing the pGSuper plasmid, and Dan Sylvester for assistancewith the preparation of this manuscript.

Correspondence should be addressed to Jelena Radulovic, 303 East Chicago Avenue, Ward 9-221, Feinberg Schoolof Medicine, Northwestern University, Chicago, IL 60611. E-mail: [email protected].

C. Gao’s present address: Department of Biochemistry, Jiangsu Key Laboratory of Brain Disease Bioinformation,Xuzhou Medical College, Xuzhou, Jiangsu, China.

DOI:10.1523/JNEUROSCI.1300-11.2011Copyright © 2011 the authors 0270-6474/11/318533-10$15.00/0

The Journal of Neuroscience, June 8, 2011 • 31(23):8533– 8542 • 8533

ERK signaling in the dendritic cytoskeleton (Schrick et al., 2007).Given that signal transduction mediated by the ERK pathway hasbeen implicated in cognitive (Chandler et al., 2001; Sweatt, 2004),emotional (Ailing et al., 2008), and motivational (Gourley et al.,2008) behavior, IQGAP1 may significantly contribute to theseprocesses by regulating ERK signaling.

Here we identify IQGAP1 as a novel interacting partner ofNR2 subunits of NMDARs that contributes to NR1/NR2A traf-ficking and NR2A-mediated ERK signaling. Using inducible orconstitutive deletion of the IQGAP1 gene, we show that IQGAP1is a key regulator of dendritic spine number in the hippocampusand lateral amygdala. Mice lacking IQGAP1 exhibited significantimpairments in memory formation and long-term potentiation(LTP) but intact anxiety- and depression-like behavior. Thesefindings unravel important roles of IQGAP1 in spine regulation,signal transduction, and NMDAR trafficking and suggest a pref-erential involvement of the NR2A/IQGAP1/ERK signaling mod-ule in cognitive versus emotional and motivational behavior.

Materials and MethodsMice. Knock-out mice lacking the IQGAP1 gene were obtained from Dr.Andre Bernards (Harvard Medical School, Boston) and backcrossed tothe C57BL/6N strain (Harlan) for nine generations. Male 4- to 6- and9-week-old IQGAP1�/� and IQGAP1�/� littermates were used for elec-trophysiological recordings and behavioral experiments, respectively.Immunoblots were performed with brain tissue of mice of different ages,as described below. Primary hippocampal neurons were prepared frompostnatal day 1 (P1) mice. All studies were approved by the Animal Careand Use Committee of Northwestern University in compliance with Na-tional Institutes of Health standards.

Hippocampal cultures and treatments. The hippocampi from IQGAP1�/�

and IQGAP1 �/� mice were isolated, dissociated, and stimulated asdescribed previously (Gao et al., 2010). Briefly, cells were plated on cov-erslips coated with poly-D-lysine in 24-well culture plates at a density of60,000 – 80,000 cells per well or six-well culture plates coated with poly-D-lysine at a density of 300,000 –350,000 cells per well and grown inNeurobasal medium containing 2 mM GlutaMax, 0.5% gentamicin, and2% B27. One-half of the medium was replaced with identical mediumevery 4 d. Under these conditions, �85% of cells were viable, �90% ofcells were neurons, and cultures could be maintained for 3 weeks. Neu-rons were cultured for 12–14 d in vitro (DIV) before stimulation andfixation. One hour before stimulation, the extracellular (perfusion orbathing) solution [containing the following (in mM): 140 NaCl, 1.3CaCl2, 5 KCl, 25 HEPES, pH 7.4, 33 glucose, 0.001 TTX, and 0.001strychnine] with 40 �M CNQX and 5 �M nimodipine was changed. Dur-ing the stimulation, the TTX, CNQX, and nimodipine were removedfrom the bath solution. To activate the NMDAR/ERK pathway, neuronswere treated with 20 �M bicuculline and 100 �M glycine for 3 min,whereas to activate the NMDAR/CamKII pathway, neurons were treatedwith 20 �M NMDA and 20 �M glycine for 3 min.

shRNA and transfection. SiRNA directed against IQGAP1 was cloned intothe pGSuper plasmid (Kojima et al., 2004) to coexpress GFP and small hair-pin RNA (shRNA) simultaneously. The sequences of four siRNA (21 nucle-otide) were 1227 5�-GGAGCTAATGAATCCTGAAGC-3� (shIQ1), 20785�-GGTACCATTACTACCACAACC-3� (shIQ2), 2398 5�-GCTTACCTG-CACTCCCATAAA-3� (shIQ3), and 4437 5�-GGAACTCATCAACGACAT-TGC-3� (shIQ4), respectively. Neurons were transfected at 11 DIV usingLipofectamine 2000 (Invitrogen). Hippocampal neurons were transfectedwith GFP-NR2A (a gift from Dr. Stefano Vicini, Georgetown University,Washington, DC) at 9–10 DIV for 4 d using Lipofectamine 2000.

Immunocytochemistry and quantitative immunofluorescence. The neu-rons were fixed with 4% formaldehyde in PBS containing 4% sucrose for15 min at room temperature (RT). Cells were permeabilized and blockedsimultaneously in PBS containing 0.1% Triton X-100 and 5% serum for1 h at RT. Primary antibodies in blocking solution were added at 4°Covernight: rabbit anti-phospho-p44/42 ERK (pERK1/2) antibody (CellSignaling Technology; 1:200), mouse anti-IQGAP1(BD Biosciences;

1:200), rabbit anti-NR2A (Millipore; 1:500), mouse anti-synaptophysin(Millipore; 1:1000), or rabbit anti-GFP (Millipore; 1:1000). After rinsingwith PBS, rhodamine-conjugated (1:500) or fluorescein-isothiocyanate(FITC)-conjugated (1:300; Vector Laboratories) secondary antibodieswere added in blocking solution for 1 h at RT. After four rinses, coverslipswere mounted using an antifading reagent.

Live hippocampal neurons expressing GFP-NR2A were first labeledwith anti-GFP (1:500) antibody for 15 min at RT. After three washes withPBS, neurons were fixed and incubated with a rhodamine-labeled sec-ondary antibody. This procedure was used to label surface NR2A. Tolabel intracellular NR2A, cells were subsequently permeabilized, blocked,and incubated with anti-GFP (1:1000) antibody overnight at 4°C, fol-lowed by an FITC secondary antibody for 1 h at RT.

Images were acquired and analyzed as described previously (Gao andWolf, 2007) using a Nikon inverted microscope, CoolSNAPEZ digitalcamera, and MetaMorph software.

Golgi staining. Animals were deeply anesthetized before being killed.The brain was fixed and stained with an FD Rapid Golgistain kit (FDNeuroTechnologies) according to the instructions.

Protein extraction and immunoblot. Cultures were harvested by scrap-ing in ice-cold buffer containing protease and phosphatase inhibitors asdescribed previously (Gao et al., 2010). The hippocampus and otherbrain regions were lysed with the same method. Protein concentrationwas determined by Bio-Rad assay. The lysates (5–20 �g/well) were sub-jected to SDS-PAGE and subsequently blotted to PVDF membranes anddeveloped as described previously (Sananbenesi et al., 2002). Four hun-dred microgram hippocampal lysates of IQGAP1�/� and IQGAP1 �/�

mice were coimmunoprecipitated by using the Catch and Release v2.0reversible immunoprecipitaion system (Millipore). Anti-pERK1/2, (Sig-ma-Aldrich; 1:5000), anti-ERK (Santa Cruz Biotechnology; 1:5000),anti-c-Fos (Santa Cruz Biotechnology; 1:500), anti-NR2A (Millipore,1:3000), anti-NR2B (Millipore; 1:500), ant-NR3A (Millipore, 1:1000),anti-NR3B (Millipore; 1:1000), anti-IQGAP1 (BD Biosciences, 1:1000),and anti-PSD-95 (Sigma-Aldrich; 1:3000) antibodies were used for immu-nodetection. For coimmunoprecipitation studies, 4 �g of antibody was usedper reaction. ImageJ software (NIH) was used for quantification.

Cell surface protein labeling and detection. Hippocampal neurons wereisolated from P1 IQGAP�/� mice and their IQGAP�/� littermates asdescribed above and cultured for 14 d. Cell surface proteins were labeledand isolated using Cell Surface Protein Isolation kit (Thermo Scientific)according to the manufacturer’s instruction. Briefly, cells were washedwith PBS and incubated at 4°C in the presence of sulfosuccinimdyl-2-(biotinamido)-ethyl-1, 3’-dithiopropionate (sulfo-NHS-SS-biotin) anddisrupted in ice-cold lysis buffer to which protease inhibitors were added(Complete; Roche) with occasional sonication. A fraction of the celllysate was saved as total protein extract. Biotinylated proteins were sep-arated by incubation with NeutrAvidin agarose for 1 h at room temper-ature, washed four times with wash buffer (with Complete proteaseinhibitors), and eluted with sample buffer at 95°C. Protein extracts wereseparated on SDS-PAGE gels and transferred to PVDF membranes. Pro-teins were detected by Western blot analysis using the Snap ID system(Millipore). Anti-NR-2A (Millipore) was diluted at 1:200, and anti-actinantibody (Santa Cruz Biotechnology) at 1:333.

Hippocampal slice preparation and recordings. Horizontal hippocam-pal slices (350 �m) were made from 4- to 6-week-old (P24 –P36)IQGAP1�/ � or IQGAP1�/� mice using a Vibratome 3000 Plus. Micewere anesthetized with a ketamine/xylazine mixture before being per-fused with ice cold sucrose-rich slicing solution (SRSS) equilibrated with95% O2/5% CO2 containing 85 mM NaCl, 2.5 mM KCl, 1.25 mMNaH2PO4, 25 mM glucose, 75 mM sucrose, 25 mM NaHCO3, 10 �M DL-APV, 100 �M kynurenate, 0.5 mM CaCl2, and 4 mM MgCl2. The mice werethen rapidly decapitated and the brains removed under ice-cold SRSS.Slices were slowly warmed to 30°C and allowed to return to room tem-perature while exchanging the SRSS for oxygenated slicing artificial CSF(ACSF) solution containing 125 mM NaCl, 2.4 mM KCl, 1.2 mMNaH2PO4, 25 mM NaHCO3, 25 mM glucose, 1 mM CaCl2, 2 mM MgCl2, 10�M DL-APV, and 100 �M kynurenate. After a 1 h incubation period, sliceswere transferred to a recording chamber and continuously perfused withoxygenated ACSF solution containing (in mM) 125 NaCl, 2.4 KCl, 1.2

8534 • J. Neurosci., June 8, 2011 • 31(23):8533– 8542 Gao et al. • IQGAP1 Regulates NR2A, Spine Density, and Memory

NaH2PO4, 25 NaHCO3, 25 glucose, 2 CaCl2 and 1 MgCl2 at 25°C. Glasselectrodes were pulled from borosilicate glass to resistances of 3– 6 M�and then filled with external ACSF recording solution. Field EPSPs(fEPSPs) were evoked by stimulating the Schaffer collaterals with a mo-nopolar glass electrode filled with ACSF at a basal stimulation frequencyof 0.05 Hz. fEPSPs were recorded in the CA1 apical dendrite layer using aglass pipette (3– 6 M�) filed with ACSF recording solution. The stimulusintensity of the first pulse was adjusted to induce an fEPSP amplitude of40% of the maximal fEPSP. Three stimulation protocols were used toinduce LTP. In each case, we initially recorded fEPSPs at the basal fre-quency for 20 min before inducing LTP. LTP was induced with (1) twotheta burst stimulations (TBSs), (2) a 100 Hz high-frequency stimulation(HFS), or (3) a single TBS preceded by a priming stimulus. TBSs con-sisted of two theta burst patterns separated by 10 s, where each theta burstconsisted of a series of 10 short 100 Hz pulse trains of five 1 ms pulses eachdelivered at a frequency of 5 Hz. HFS consisted of a single 100 Hz train(100 stimuli, 10 ms each) for a 1 s duration. Priming-induced LTP con-sisted of two priming stimuli at 10 Hz for 10 s separated by 20 s, followedby an interval of 10 min of stimulation at basal frequency, and then asingle theta burst stimulation. fEPSPs were recorded for 120 min afterLTP induction in the TBS and HFS experiments, whereas the priming-induced plasticity was measured at 60 min after induction. The amountof LTP was determined by normalizing the averaged fEPSP slope duringthe last 20 min of each experiment to the 20 min of basal control. fEPSPwaveforms were acquired with pClamp 10.2 software (Molecular De-vices) at a sampling rate of 20 kHz and filtered at 1 kHz. The initial slopeand peak amplitude were measured and analyzed with Origin 7.0(OriginLab). Values are represented as the mean � SEM, and n valuesrepresent the number of recordings from individual slice preparations.Only a single recording was taken from each slice preparation. All elec-

trophysiological recordings and analyses were performed in a blind fash-ion. For presentation purposes, data were normalized and averaged inone-minute bins. However, all statistical analyses were performed on theraw data set.

Fear conditioning. Contextual and tone-dependent fear conditioningwas performed in an automated system (TSE Systems) and consisted of asingle exposure to a context (3 min) followed by a 30 s tone (10 kHz; 75dB SPL) and a foot shock (2 s; 0.7 mA; constant current) as describedpreviously (Radulovic et al., 1998). Context-dependent freezing wasmeasured 24 h later every 10th second over 180 s by two observers un-aware of the experimental conditions and expressed as percentage of totalnumber of observations. Freezing to the tone was scored every fifth sec-ond in a novel context during a 30 s exposure.

Object discrimination. Animals were habituated for 5 min in an 80 � 80cm arena on day 1. During the following 2 consecutive days, mice wereexposed for 5 min to two different objects, object 1 (O1) and object 2(O2), placed in two neighboring quadrants. On test day 4, O1 remainedat the same location, whereas O2 was either moved to a new locationor replaced by a new object [object 3 (O3)]. The percentage of timethat mice spent visiting O1, O2, or O3 was measured during the 5 minexposure.

Anxiety. Anxiety-like behavior was evaluated using the dark/light(D/L) emergence task. A 10 � 10 � 10 cm shelter was placed in themiddle of the 80 � 80 cm arena. Latency to come out from the shelter andtime spent in the dark area during a 5 min test were recorded automati-cally by VideoMot (TSE Systems).

Depression. For the novelty-suppressed feeding test, mice were de-prived of food overnight and water for 3 h before testing, sufficient toproduce a state of mild hunger but little discomfort. For the testingexposure, mice were placed in a brightly lit open field (80 � 80 cm)

Figure 1. IQGAP1 is abundant in the brain but its deficiency does not affect CaMKII signaling, Ras-GTPase activity, or levels of N-cadherin and �-catenin. A, IQGAP1 in cultured hippocampalneurons derived from wild-type mice, as detected by immunocytochemistry from DIV 3–21. B, Immunoblot showing the IQGAP1 levels in the hippocampus of wild-type mice of different postnatalage. C, Quantification of the immunoblots reveals a drop of IQGAP1 level after postnatal day 3 but steady levels thereafter. D, IQGAP1 signals were absent in hippocampal extracts (left) and neurons(right) of IQGAP1�/� mice. E, IQGAP1 signals were strong in the hippocampus, cortex, cerebellum, and striatum of 9-week-old IQGAP1 �/� mice but lacking in their IQGAP1�/� littermates. F,Immunoblots showing similar levels of pCaMKII or CaMKII in the hippocampus of IQGAP1 �/� and IQGAP1�/� mice. Coimmunoprecipitations (IP) of pCaMKII and IQGAP1 confirmed the interactionof these proteins in wild-type mice and its absence in IQGAP1 knock-outs (right). Stimulation (Stim) of NMDAR significantly increased the level of pCaMKII versus control (Cont) in both genotypes(middle). Quantification of the fold pCaMKII/CaMKII change of control (right) is shown (n � 3–5; *p 0.05 when compared to unstimulated Cont). Data are represented as mean � SEM. G, Raspull-down test showed similar Ras-GTPase activity in hippocampal extracts of IQGAP1�/� when compared to IQGAP1 �/� mice. H, Similar levels of N-cadherin and �-catenin in wild-type andIQGAP1 knock-out mice as revealed by immunoblot. Scale bars: A, 20 �m; D, 20 �m. OD, Optical density.

Gao et al. • IQGAP1 Regulates NR2A, Spine Density, and Memory J. Neurosci., June 8, 2011 • 31(23):8533– 8542 • 8535

enclosed by 20 cm walls. A single food pelletwas placed in the center of the field and a videotracking system monitored the patterns offeeding and exploration during the 5 min trial.Latency to approach the food was recorded.For the forced swim test, mice were placed inan upright cylinder (20 cm diameter) filledwith lukewarm water (26°C) up to 5 cm belowits opening. Mice were observed for 6 min, andthe amount of time spent in an immobile pos-ture during the last 5 min was scored.

The experimenters performing the behav-ioral assays were always blind to genotype,and most behavioral measures were capturedautomatically.

Statistical analyses. Statistical comparisonsbetween groups were performed by two-tailedmultiple t test with Bonferroni’s correction.

ResultsIQGAP1 is abundant in the CNS, butIQGAP1 deletion does not affect grossneuronal functionIn primary hippocampal cultures, IQGAP1was mainly detected in the soma on DIV 3,but by DIV 21 it also became abundantin neuronal processes (Fig. 1 A). In hip-pocampal lysates, a strong IQGAP1-specificband at the expected size of 190 kDa wasobserved at different postnatal ages (Fig.1 B). Quantification revealed a decreaseof IQGAP1 level after day 3 and steadylevels afterward (Fig. 1C). Culturesfrom IQGAP1 knock-out mice did notshow specific signals for this protein byimmunoblot (Fig. 1D, left) or immuno-fluorescence (right). We did not noticeany obvious deficits of neuronal polarityin the cultures of IQGAP1 knock-outs.In lysates of different brain regions ofadult wild-type but not knock-out mice,IQGAP1 was expressed in the hippocam-pus, cortex, cerebellum, and striatum(Fig. 1E). Despite the disrupted interac-tion between IQGAP1 and CaMKII in

Figure 2. Downregulation of ERK signaling in IQGAP1�/� mice. A, Hippocampal neurons derived from IQGAP1 �/� or IQGAP1�/� mice were treated with bicuculline (20 �M) and glycine (100�M) for 3 min to activate Syn NMDARs. IQGAP1 and pERK colocalize in stimulated IQGAP1 �/� neurons. Double-label immunostaining for IQGAP1 (red, top), pERK1/2 (green, middle), and merge(yellow, bottom) revealed significantly reduced level of pERK1/2 in IQGAP1�/� neurons. Scale bar, 20 �m. B, Representative immunoblots showing decreased pERK1/2 (normalized to total ERK1/2)levels after stimulation (Stim) of NMDARs in IQGAP1�/� neurons with bicuculline and glycine. Quantification of fold change of immunoreactivity (pERK/ERK) from unstimulated control (Cont) ispresented below (n � 3; *p 0.05 compared to corresponding IQGAP1 �/�). C, Representative immunoblots showing decreased c-fos versus actin levels in IQGAP1�/� versus IQGAP1 �/�

neurons. Quantification of the fold change of immunoreactivity (c-fos/actin) from control is presented below (n � 3; *p 0.05 compared to IQGAP1 �/�). D, Representative immunoblots showingdecreased pERK1/2 in the adult hippocampus after fear conditioning (FC) of IQGAP1�/� versus IQGAP1 �/� mice. Quantification of the fold change of immunoreactivity (pERK2/ERK2) is presentedbelow (n � 4; ***p 0.001 compared to IQGAP1 �/�). E, The levels of c-fos were also reduced as revealed by decreased c-fos versus actin (representative immunoblots; top) and quantificationof the fold change of immunoreactivity (c-fos/actin; bottom; n � 3– 4; *p 0.05 compared to IQGAP1 �/�). Data are represented as mean � SEM.

Figure 3. IQGAP1 interacts with NR2 subunits and regulates their surface expression. A, IQGAP1 interacted with NR2A, and to a lesserextent NR2B (top) but not NR3A or NR3B (bottom). B, IQGAP1 also interacted with PSD-95. C–E, Representative immunoblots showing thelevels of NR1 (C), NR2A (D), and NR2B (E) in membrane fractions derived from IQGAP1 �/� and IQGAP1�/� mice. The ratios of NR/actinwere quantified and presented below the corresponding immunoblots. Membrane levels of NR1 and NR2A were significantly reduced inIQGAP1�/� versus IQGAP1 �/� membranes (n � 3– 4; *p 0.05 compared to IQGAP1 �/�). F, Double staining of surface NR2A inhippocampal neurons (left). Analyses of dendritic surface (red), intracellular (green), and total (red and green) NR2A reveal reduced surfaceNR2A expression in IQGAP1 knock-outs (right). G, Quantification of GFP-NR2A surface intensity (left), GFP-NR2A total intensity (middle),and the ratio of surface/total GFP-NR2A (right; n � 8 for IQGAP1�/�, 12 for IQGAP1�/� neurons. H, Biotinylation assays revealed asignificant reduction of the ratio between surface (S) and total (T) endogenous NR2A in cultured hippocampal neurons (left, immunoblot;right, quantification). Total NR2A levels were determined in input lysates whereas surface NR2A were determined after elution from theavidin column (the total band thus appears weaker because the sample is more dilute). **p 0.01; ***p 0.001 compared to IQ-GAP1 �/�. Scale bar: F, left, 20 �m; right, 5 �m. Data are represented as mean � SEM.


IQGAP1 knock-outs, CaMKII phosphorylation remained intact(Fig. 1F). Similarly, Raf1-dependent Ras activity (Fig. 1G) andN-cadherin/�-catenin levels (Fig. 1H) were similar in IQGAP1knock-outs and their wild-type littermates.

Impaired neuronal signal transduction in IQGAP1�/� miceContrary to the other signaling pathways, ERK activation wasmarkedly impaired under in vitro and in vivo conditions thattypically trigger the ERK/c-fos pathway. In cultured hippocampalIQGAP1�/� neurons, NMDAR stimulation with bicuculline andglycine failed to induce ERK phosphorylation (p 0.05) (Fig.2A,B) and c-Fos production when compared to their wild-typelittermates (p 0.05) (Fig. 2C). Similarly, in hippocampi ofIQGAP1 knock-outs, fear conditioning did not induce upregula-tion of phosphorylated ERK (pERK; p 0.001) (Fig. 2D) or c-fos(p 0.05) (Fig. 2E). These results suggest that IQGAP1 plays acritical role in ERK signaling.

Impaired NR2A-mediated ERK signaling and NR2A surfaceexpression in IQGAP1�/� neuronsERK/c-fos signaling in response to NMDAR stimulation is pre-dominantly mediated by the NR2A subunit (Gao et al., 2010). Wetherefore hypothesized that the IQGAP1 knock-out affects thefunction of NR2A. Both NR2A and NR2B, but not NR3, coim-munoprecipitated with IQGAP1 (Fig. 3A). However, NR2A/IQGAP1 signals were consistently stronger than NR2B/IQGAP1

signals across three replicates. IQGAP1also associated with PSD-95 (Fig. 3B), akey scaffolding protein of NMDARs. Wenext examined whether the traffickingof NMDAR subunits was affected inIQGAP1 knock-outs. Membrane levels ofNR1 (Fig. 3C) and NR2A (Fig. 3D) weresignificantly decreased in IQGAP1�/�

when compared to IQGAP1 �/� mice(p 0.05), whereas NR2B levels were notchanged (Fig. 3E). The surface expressionof NR2A was also examined using hip-pocampal neurons transfected with GFP-tagged NR2A at the N terminus (Luo etal., 2002). The levels of surface GFP-NR2A and the ratio between surface andtotal GFP-NR2A were significantly de-creased (p 0.001) in IQGAP1�/� com-pared to IQGAP1 �/� cultures (Fig.3F,G). Similarly, a reduction of endoge-nous surface NR2A, revealed as a signifi-cantly lower (p 0.001) ratio of surfaceversus total NR2A (Fig. 3H), was demon-strated using a biotinylation assay. Theseexperiments identify NR2A as a novel in-teracting partner of IQGAP1. Moreover,this interaction seems to be highly rele-vant for the regulation of surface expres-sion of NR2A-containing NMDARs inhippocampal neurons.

Region-specific decrease of spinenumbers in IQGAP1�/� miceBy regulating the cytoskeleton of neuro-nal cells, IQGAP1 contributes to neuronaloutgrowth (Li et al., 2005) and promotesspine formation. Accordingly, inducible

downregulation of IQGAP1 with shIQGAP14 (shIQ4, an shRNAthat reduced the level of IQGAP1 by 48%) (Fig. 4A) signifi-cantly decreased spine density when compared to the pGSupervector control (p 0.001) (Fig. 4B). Hippocampal neurons incultures prepared from IQGAP1�/� mice also exhibited lowerspine density than IQGAP�/� neurons (p 0.001) (Fig. 4C).

In the adult brain, IQGAP1 knock-out also resulted in reducedspine numbers, as revealed by Golgi staining; however, the effectwas region specific. Significantly lower spine densities were foundin the hippocampus (Fig. 4D) (p 0.001) and lateral amygdala(p 0.01), but not medial prefrontal cortex or basal amygdala ofIQGAP1 knock-outs when compared to their wild-type litter-mates (Fig. 4E). Together, these findings show that inducible andconstitutive IQGAP1 deficiency results in enduring reduction ofdendritic spine density.

Impaired LTP of IQGAP1 knock-outs in response toweak stimuliWe next tested whether the marked effects of IQGAP1 deletionon NR2A distribution and ERK signaling were reflected in alteredNMDAR-dependent LTP in acute hippocampal slice prepara-tions. Input– output relationships for fEPSPs evoked by stimula-tion of Schaffer collateral–CA1 pyramidal cell synapses recordedfrom wild-type and IQGAP1 knock-outs were equivalent (Fig.5A). Three LTP induction paradigms were used to comparefEPSP potentiation in response to both strong and weak stimu-

Figure 4. Region-specific decrease of spine density after deletion of IQGAP1. A, Human embryonic kidney 293 cells weretransfected for 3 d with pGSuper (express GFP) and shIQGAP1 (shIQ) that coexpress both GFP and siRNA against IQGAP1. Down-regulation of IQGAP1 was achieved by shIQ4. B, Spine density in IQGAP1�/� neurons transfected with pGSuper or shIQ4 and inIQGAP1 knock-outs. Representative immunostainings for GFP (top) and quantification of spine number (bottom) revealed asignificant decrease of spine number by shIQ4 and IQGAP1 knock-out (n � 23 pGSuper neurons, 29 shIQ4 neurons; ***p 0.001,shIQ4 compared to pGSuper control). Quantification of spine number also showed a significant reduction in IQGAP1�/� (***p 0.001) when compared to IQGAP1�/� neurons neurons (n � 22 IQGAP1 �/� and 15 IQGAP1�/� neurons). C, Golgi stain of thehippocampus and outline of the areas used for spine analyses. D, Reduced spine density in the hippocampus and lateral amygdala(LA) of 9-week-old IQGAP1 �/� and IQGAP1�/� mice (***p 0.001 compared to corresponding IQGAP1 �/� littermates). E,Intact spine density in the medial prefrontal cortex (mPFC) and basal amygdala (BA) of the same mice. The numbers of analyzeddendrites were 24 IQGAP1 �/� and 27 IQGAP1�/� for hippocampus, 22 IQGAP1 �/� and 23 IQGAP1�/� for mPFC, 25 IQ-GAP1 �/� and 29 IQGAP1�/� for the LA, and 16 IQGAP1 �/� and 20 IQGAP1�/� for the BA. Scale bars: B, 5 �m; C, left panels,100 �m; right panels, 50 �m; D, E, 5 �m. Data are represented as mean � SEM..


lation. Slices from IQGAP1 mice exhib-ited similar levels of TBS LTP as theirwild-type counterparts (fEPSP potentia-tion, 133 � 10% for wild-type, 142 � 16%for knock-outs; n � 11 and 13 slices,respectively) (Fig. 5B). Similarly, LTPinduced by high-frequency tetanic stim-ulation was equivalent in wild-type andknock-out mice (121 � 6% and 132 �11%, respectively; n � 9 slices each) (Fig.5C). In contrast, a single theta burst traindelivered 10 min after an initial primingstimulation elicited potentiation in re-cordings from wild-type slices (128 � 9%;n � 6) but was not induced in IQGAP1knock-out slices (92 � 12%; p 0.05)(Fig. 5D). These results suggest that cellu-lar plasticity induced by a weak stimula-tion of excitatory synapses on CA1pyramidal neuron is selectively abrogated inthe IQGAP1�/� mice, whereas more robustforms of plasticity remain intact.

Memory deficits in IQGAP1�/� miceIQGAP1 knock-outs had a significant deficitin contextual fear conditioning (t � 3.346;p 0.01) (Fig. 6A), tone-dependent fearconditioning (t�2.375; p0.05) (Fig. 6B),and discrimination between the fear condi-tioning and novel contexts (wild type, t �3.946, p 0.01 vs context 1; knock-out, t �0.144, p � 0.888 vs context 1) (Fig. 6C)when compared to their wild-type litter-mates. Lack of IQGAP1 did not affect ac-tivity in response to context (t � 1.089; p � 0.297) or electricshock (t � 0.785; p � 0.447) (Fig. 6D,E).

In the object recognition/discrimination paradigm, bothIQGAP1�/� and IQGAP1�/� mice showed similar exploration ofnovel objects 1 and 2 during training (wild type, t � 0.121, p �0.905, O2 vs O1; knock-out, t � 0.099, p � 0.922, O2 vs O1) (Fig.6F). During testing, IQGAP1 wild types spent significantly moretime with the object whose location was changed (t � 2.593; p 0.05 vs O1) (Fig. 6G), whereas IQGAP1 knock-outs did not (t �0.298; p � 0.772). Similarly, IQGAP1 wild types spent signifi-cantly more time with a novel object 3 (t � 4.400; p 0.001 vsO1) (Fig. 6H), whereas IQGAP1 knock-outs equally explored thefamiliar (O1) and novel (O3) objects (t � 1.459; p � 0.175). Thiswas not because of differences in activity in response to O3 (t �0.099; p � 0.922) (Fig. 6 I). There were also no changes in grossneuronal morphology or body weight (data not shown), indicat-ing that developmental or sensory–motor deficits did not con-tribute to the observed memory impairments. Most of theseimpairments were reproduced in female mice and could be over-come by massed training (data not shown). Together, IQGAP1knock-outs show impairments in contextual, spatial, cued, andobject learning tasks.

Normal anxiety- and depression-like behavior in IQGAP1�/�

miceWe next examined whether deletion of IQGAP1 affects anxiety ordepression-like behavior. These behaviors were evaluated usingthe dark/light emergence, novelty-suppressed feeding, and forcedswim tests. In the D/L test, there were no significant differences be-

tween IQGAP1�/� and IQGAP1�/� mice in the latency to exit (t �0.602; p � 0.553) (Fig. 7A) and percentage of time spent in the darkarea (t � 1.441; p � 0.163) (Fig. 7B). In the novelty-suppressedfeeding test, the latency to eat the food pellet placed in the center ofan open field was similar in IQGAP1�/� and IQGAP1�/� mice (t �0.766; p � 0.456) (Fig. 7C). Similarly, the time spent in an immobileposture in the forced swim test was not significantly different be-tween IQGAP1�/� and IQGAP1�/� mice (t � 0.541; p � 0.596)(Fig. 7D). These results demonstrate that deletion of IQGAP1 doesnot affect anxiety or depression-like behaviors.

DiscussionWe showed that IQGAP1 is required for ERK signaling, region-specific dendritic spine density, synaptic plasticity, and memory.We also demonstrated that IQGAP1 interacts with PSD-95 andNR2A and regulates the surface expression of NR2A on hip-pocampal neurons. Some of the observed molecular, cellular, andbehavioral effects of the IQGAP1 knock-out may thus be medi-ated by the newly described interactions between IQGAP1, PSD-95, and NR2 subunits. Surprisingly, motivational and emotionalbehaviors remained intact. On the basis of these findings, wepropose that IQGAP1 plays a nonredundant role in a subset ofmechanisms underlying memory formation.

In various cells and tissues, IQGAP1 has primarily been rec-ognized as a scaffolding protein for the cadherin/catenin, cal-modulin, Rac1/Cdc42, and MEK/ERK pathways (Brown andSacks, 2006; Johnson et al., 2009). Nevertheless, the peripheralconsequences of IQGAP1 deletion, in particular those associatedwith cadherin function and development, were minimal (Li et al.,

Figure 5. IQGAP1�/� mice exhibit a deficit in cellular plasticity in response to weak induction stimuli. A, Input– output curvesfor CA1 fEPSPs evoked by Schaffer collateral stimulation are equivalent in wild-type and IQGAP1�/� mice. B, Theta burst LTP wasequivalent in wild-type and IQGAP�/� mice. Plots show the averaged data over all the recordings, with the black arrow indicatingwhen TBS (as defined in Materials and Methods) was induced in the experiments (n � 11 and 13 slices from each genotype,respectively). C, LTP induced by HFS was equivalent in wild-type and IQGAP�/� fEPSP recordings (n � 9 slices from eachgenotype). D, LTP induced by priming a single theta burst with two 10 Hz stimulations induces LTP of fEPSPs in wild-type slices butnot in IQGAP�/� hippocampal slices (n � 6 slices from each genotype; p 0.05). Data are represented as mean � SEM.


2000). In the brain, the levels, distribution, and activationpatterns of most IQGAP1-regulated pathways were similarlyunaffected in IQGAP1 knock-outs, as were gross neuronalmorphology, maturation, and sensory–motor behavior. Thismay be attributable to the constitutive lack of IQGAP1 triggeringcompensatory processes or, alternatively, to regulatory redun-dancy of cadherin/catenin, CaMKII, and Rac1/Cdc42 signalingby other proteins and scaffolds. A notable exception, however,was the ERK pathway, which exhibited a significantly impairedphosphorylation. Activation and nuclear propagation of ERKsignaling is robustly induced by stimulation of NMDAR in vitro(Ivanov et al., 2006; Gao et al., 2010) or learning processes in vivo(Sananbenesi et al., 2002; Shalin et al., 2006; Schrick et al., 2007),and is predominantly mediated by the NR2A subunit (Gao et al.,2010). However, in the IQGAP1 knock-outs, both experimentalconditions failed to trigger ERK phosphorylation despite normal

ERK levels, possibly because of impaired NR2A trafficking, sur-face expression, or ineffective scaffolding to ERK.

IQGAP1 interacted with NR2A and PSD-95, to a lesser extentwith NR2B, and not with NR3 subunits, which are known fortheir lack of binding to PSD-95 (Eriksson et al., 2007). This iden-tifies IQGAP1 as a component of NR1/NR2/PSD-95 complexes,regulating NR2A trafficking and NR2A-induced ERK signaling.Given the key involvement of this NMDAR subunit in long-termmemory formation, (Kishimoto et al., 1997; Sprengel et al., 1998),the role of IQGAP1 in cognition may be mediated by its interac-tion with NR2A. It should be noted, however, that NR2A havealso been implicated in anxiety and depression (Boyce-Rustayand Holmes, 2006), two behaviors that remained unaffected inthe IQGAP1 knock-out. Our findings thus suggest that anx-iogenic and depressant effects of NR2A do not critically dependon IQGAP1/ERK interactions and are likely mediated by differ-ent scaffolding or signaling complexes.

Despite the ubiquitous distribution of IQGAP1 in the CNS,the decrease in spine numbers in IQGAP1 knock-out mice wasregion specific, with the hippocampus and lateral amygdala mostaffected. This neuroanatomical specificity resulting from a gen-eral genetic deficit is reminiscent of an earlier observation inmutant mice lacking kalirin, an important regulator of spine den-sity and morphology (Cahill et al., 2009). These localized effectslikely depend on the regional repertoire of functionally relatedmolecular pathways that compensate for the deficiency of theknock-out. In the present study, it is very likely that regionalactions of IQGAP2 or IQGAP3 overcame the IQGAP1 deficit,given the structural homology and functional redundancy ofthese family members. All IQGAP proteins share the ability tointeract with actin filaments, a feature that is particularly impor-tant for their redundant effects on neuronal outgrowth (Fukata etal., 1997; Wang et al., 2007), and most likely spine formation, asobserved in our study. Interestingly, whereas the involvement ofIQGAP1 (but not IQGAP2 and IQGAP3) in the growth of hip-pocampal axons was dispensable (Wang et al., 2007), its role inhippocampal dendritic spine density observed here was nonre-dundant. Thus, in addition to regional specificity, IQGAP pro-

Figure 6. IQGAP1 deletion causes memory deficits. Impaired fear conditioning and context discrimination in IQGAP1�/� mice when compared to IQGAP1 �/� littermates. A, Context-dependentfear conditioning (**p 0.01; n � 8 for IQGAP1 �/�; n � 10 for IQGAP1�/�). B, Tone-dependent fear conditioning (*p 0.05; n � 8 for IQGAP1 �/�; n � 10 for IQGAP1�/�). C, Freezing tocontext 1 where shock was delivered during training versus freezing to a novel context 2 (n � 9 for IQGAP1�/�; n � 7 for IQGAP1�/�; **p 0.01 when compared to context 1). D, Similarlocomotor activity of IQGAP1 �/� mice and IQGAP�/[minus ] mice (n � 7 for IQGAP1 �/�; n � 8 for IQGAP�/�). E, Similar shock responses were recorded for IQGAP1 �/� and IQGAP1�/� mice(n � 7 for IQGAP1 �/�; n � 8 for IQGAP1�/�). F, No preference for object 1 (black) or object 2 (gray) during training. G, Impaired object discrimination in IQGAP1�/� mice when compared toIQGAP1�/ � littermates as revealed by a similar percentage of visiting time for objects 1 and 2 when object 2 is presented in a new spatial location. H, Impaired object discrimination in IQGAP1�/�

mice when compared to IQGAP1 �/� littermates as revealed by similar percentage of visiting time for object 1 and a novel object 3 (n �9 for IQGAP1 �/�; n �7 for IQGAP1�/�). For G and H, *p 0.05, ***p 0.001 compared to object 1. I, Similar exploration of objects 1, 2, and 3 by IQGAP�/� mice (n � 7 per group). Data are represented as mean � SEM.

Figure 7. IQGAP1 deletion does not affect anxiety- or depression-like behavior. A, B, Dark/light test showing similar latency to exit the dark area (A) and the percentage of time spent indark area (B) in IQGAP1�/� and IQGAP1 �/� mice; n �12 (IQGAP1 �/�) and 13 (IQGAP1�/�). C,Novelty-suppressed feeding test showing similar latency to start consumption of food pelletslocated in the center of an open field of IQGAP1�/� and IQGAP1 �/� mice; n � 6(IQGAP1 �/�) and 10 (IQGAP1�/�). D, Forced swim test showing similar time spent floating inIQGAP1�/� versus IQGAP1 �/� mice; n � 8 (IQGAP1 �/�) and 10 (IQGAP1�/�). Data arerepresented as mean � SEM.


teins may exert subcellular specificity in their actions. A detailedanalysis of the neuroanatomical and cellular distribution ofIQGAP proteins is critical for better understanding of theirunique and redundant roles.

Comparative analysis of cellular plasticity in wild-type andIQGAP1 knock-out mice supports a role for the scaffolding pro-tein in transducing weak but not strong stimuli into stable poten-tiation of CA1 excitatory transmission. Long-term plasticity wasinduced using three distinct models: a single TBS preceded bytwo 10 Hz priming trains, two trains of TBS, or a strong 100 Hzstimulus. LTP of CA1 fEPSPs occurred after the two strongerinduction paradigms but was absent in hippocampi fromIQGAPI mice after the single primed TBS. In principle, the lack ofpotentiation in this model could result from signaling deficitswithin the priming phase or simply as a result of the weaker(single TBS) induction stimulus compared to the other para-digms tested. TBS with a prior 10 Hz stimulation is permissive forNMDA-receptor-dependent LTP through complex homosynap-tic and heterosynaptic pathways in which mGlu receptors playcrucial roles (Chevaleyre and Castillo, 2004; Abraham, 2008).Postsynaptic priming follows activation of group I mGlu recep-tors, which enhances CA1 pyramidal cell excitability and facili-tates induction of LTP (Cohen et al., 1999; Abraham, 2008).Heterosynaptic metaplasticity, in contrast, occurs through de-pression of inhibitory tone on pyramidal neurons subsequent tomGlu-receptor-driven synthesis of endocannabinoids, which ac-tivate cannabinoid receptors on interneuron presynaptic termi-nals and consequently reduce release of GABA (Chevaleyre andCastillo, 2004). Ablation of IQGAP1 might interfere with a vari-ety of elements of these metaplastic signaling pathways, or alter-natively, NMDA receptor signaling might be weakened in thehippocampus of IQGAP1 knock-outs such that threshold induc-tion stimuli of any sort no longer evoke potentiation. In line withthis view, the specific roles of individual scaffolding proteins inthe regulation of LTP are thought to depend on their contribu-tion to unique NMDAR signaling complexes (Carlisle et al.,2008). Stronger stimulation paradigms clearly elicited LTP in theIQGAP knock-out mice, as also seen in several studies using spe-cific and limited functional disruption of the NMDAR scaffoldsPSD-95 and SynGAP1 (Lim et al., 2003; Rama et al., 2008). Anal-ogous to the observed effects of IQGAP1 knock-out on neuronalplasticity, we found marked learning deficits with one-trial trainingprotocols in the associative-conditioning and object-recognitionparadigms that were overridden by intensified training. These find-ings suggest that stronger stimuli recruit additional, redundant mo-lecular mechanisms that are able to compensate for the observedLTP and learning deficits.

General or brain-region-specific decreases in spine densityaccompany major neuropsychiatric disorders, such as schizo-phrenia (Glantz and Lewis, 2000), major depression (Law et al.,2004), and mental retardation (Fiala et al., 2002). Commonly,deletion of a single gene results in reduction of spine density andmultiple behavioral phenotypes (Cahill et al., 2009; Ayhan et al.,2010). It thus remains unclear why the IQGAP1 knock-outs se-lectively exhibited memory deficits. Reduced spine numbers inhippocampus and lateral amygdala, along with decreased ERKsignaling in the hippocampus, could provide a neuroanatomicalbasis for selective cognitive deficits of contextual fear condition-ing (Kim and Fanselow, 1992; Phillips and LeDoux, 1992), objectrecognition memory (Kelly et al., 2003; Fernandez et al., 2008),and tone-dependent fear conditioning (Schafe et al., 2000; Radleyet al., 2006; Lamprecht et al., 2009). Alternatively, spine densitymay be specifically involved in the regulation of mnemonic

rather than emotional and motivational processes, and changesseen in mood disorders (Li et al., 2010) could be related to thecognitive deficits accompanying these illnesses.

Contrary to NR2A, IQGAP1 did not seem to play a major rolein NR2B function. Alternatively, preferential NR2B scaffolds,such as SynGAP1 (Kim et al., 2005), RACK1 (Yaka et al., 2003),SAP102 (Petralia et al., 2005), or DAPK1 (Tu et al., 2010), mighthave compensated for the loss of IQGAP1. However, this is un-likely given that the functional consequences of IQGAP1 deletionseemed opposite from those caused by NR2B/PSD-95 scaffolds.IQGAP1 knock-out resulted in a significant impairment of ERKsignaling, in accordance with a positive MEK/ERK regulatoryrole of IQGAP1 (Brown and Sacks, 2006) and NR2A (Kim et al.,2005). On the other hand, lack of SynGAP1, a functionally relatedscaffold of the GAP protein family, increases the spine numberand accelerates spine maturation in primary hippocampal cul-tures (Vazquez et al., 2004). Furthermore, reduced levels of Syn-GAP1 increase ERK phosphorylation (Komiyama et al., 2002),consistent with the negative regulation of the ERK pathway bySynGAP (Rumbaugh et al., 2006) and NR2B (Kim et al., 2005). Itis possible that distinctive functions of GAP domains account fordecreased ERK signaling by NR2B/SynGAP1 versus enhancedERK signaling by NR2A/IQGAP1 complexes: SynGAP1 acceler-ates hydrolysis of bound GTP on Ras, whereas IQGAP1, using itsGAP-related domain, binds to Cdc42 and Rac1 and stabilizestheir GTP-bound states (Kurella et al., 2009). The different mo-lecular and morphological actions of IQGAP1 and SynGAP1probably mediate their discrete effects on behavior: unlike Syn-GAP1 deficiency, which affects working and reference memory,activity, anxiety, and object recognition memory (Muhia et al.,2010), IQGAP1 knock-out caused profound deficits of object rec-ognition and other forms of memory without alterations of mo-tivational or emotional behavior. These findings stress theimportance of scaffolding proteins as generators of stimulus-specific neuronal signaling patterns (Sheng and Kim, 2002) un-derlying distinctive behavioral profiles.

Together, we identified IQGAP1 as a component of NR2A/PSD-95 complexes playing a principal role in NR2A trafficking,ERK signaling, dendritic spine density, and memory. Given thatregion-specific alterations of dendritic spine morphology andmemory deficits, as found during IQGAP1 deficiency, are com-monly observed in Alzheimer’s and other types of dementia(Aoki et al., 2007; Kasai et al., 2010; Schulz-Schaeffer, 2010),IQGAP1 malfunction may have significant implications in cog-nitive symptoms of human psychiatric disorders.

ReferencesAbraham WC (2008) Metaplasticity: tuning synapses and networks for plas-

ticity. Nat Rev Neurosci 9:387.Ailing F, Fan L, Li S, Manji S (2008) Role of extracellular signal-regulated

kinase signal transduction pathway in anxiety. J Psychiatr Res 43:55– 63.Aoki C, Mahadomrongkul V, Fujisawa S, Habersat R, Shirao T (2007)

Chemical and morphological alterations of spines within the hippocam-pus and entorhinal cortex precede the onset of Alzheimer’s disease pa-thology in double knock-in mice. J Comp Neurol 505:352–362.

Ayhan Y, Abazyan B, Nomura J, Kim R, Ladenheim B, Krasnova IN, Sawa A,Margolis RL, Cadet JL, Mori S, Vogel MW, Ross CA, Pletnikov MV(2011) Differential effects of prenatal and postnatal expressions of mu-tant human DISC1 on neurobehavioral phenotypes in transgenic mice:evidence for neurodevelopmental origin of major psychiatric disorders.Mol Psychiatry 16:293–306.

Boyce-Rustay JM, Holmes A (2006) Genetic inactivation of the NMDA re-ceptor NR2A subunit has anxiolytic- and antidepressant-like effects inmice. Neuropsychopharmacology 31:2405–2414.

Brandt DT, Grosse R (2007) Get to grips: steering local actin dynamics withIQGAPs. EMBO Rep 8:1019 –1023.


Briggs MW, Sacks DB (2003) IQGAP proteins are integral components ofcytoskeletal regulation. EMBO Rep 4:571–574.

Brown MD, Sacks DB (2006) IQGAP1 in cellular signaling: bridging theGAP. Trends Cell Biol 16:242–249.

Cahill ME, Xie Z, Day M, Photowala H, Barbolina MV, Miller CA, Weiss C,Radulovic J, Sweatt JD, Disterhoft JF, Surmeier DJ, Penzes P (2009) Ka-lirin regulates cortical spine morphogenesis and disease-related behav-ioral phenotypes. Proc Natl Acad Sci U S A 106:13058 –13063.

Carlisle HJ, Fink AE, Grant SG, O’Dell TJ (2008) Opposing effects ofPSD-93 and PSD-95 on long-term potentiation and spike timing-dependent plasticity. J Physiol 586:5885–5900.

Chandler LJ, Sutton G, Dorairaj NR, Norwood D (2001) N-methyl-D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures. J Biol Chem276:2627–2636.

Chevaleyre V, Castillo PE (2004) Endocannabinoid-mediated metaplastic-ity in the hippocampus. Neuron 43:871– 881.

Cohen AS, Coussens CM, Raymond CR, Abraham WC (1999) Long-lastingincrease in cellular excitability associated with the priming of LTP induc-tion in rat hippocampus. J Neurophysiol 82:3139 –3148.

Eriksson M, Samuelsson H, Samuelsson EB, Liu L, McKeehan WL, BenedikzE, Sundstrom E (2007) The NMDAR subunit NR3A interacts withmicrotubule-associated protein 1S in the brain. Biochem Biophys ResCommun 361:127–132.

Fernandez SM, Lewis MC, Pechenino AS, Harburger LL, Orr PT, Gresack JE,Schafe GE, Frick KM (2008) Estradiol-induced enhancement of objectmemory consolidation involves hippocampal extracellular signal-regulated kinase activation and membrane-bound estrogen receptors.J Neurosci 28:8660 – 8667.

Fiala JC, Spacek J, Harris KM (2002) Dendritic spine pathology: cause orconsequence of neurological disorders? Brain Res Brain Res Rev39:29 –54.

Fukata M, Kuroda S, Fujii K, Nakamura T, Shoji I, Matsuura Y, Okawa K,Iwamatsu A, Kikuchi A, Kaibuchi K (1997) Regulation of cross-linkingof actin filament by IQGAP1, a target for Cdc42. J Biol Chem272:29579 –29583.

Gao C, Wolf ME (2007) Dopamine alters AMPA receptor synaptic expres-sion and subunit composition in dopamine neurons of the ventral teg-mental area cultured with prefrontal cortex neurons. J Neurosci27:14275–14285.

Gao C, Gill MB, Tronson NC, Guedea AL, Guzman YF, Huh KH, CorcoranKA, Swanson GT, Radulovic J (2010) Hippocampal NMDA receptorsubunits differentially regulate fear memory formation and neuronal sig-nal propagation. Hippocampus 20:1072–1082.

Glantz LA, Lewis DA (2000) Decreased dendritic spine density on prefron-tal cortical pyramidal neurons in schizophrenia. Arch Gen Psychiatry57:65–73.

Gourley SL, Wu FJ, Kiraly DD, Ploski JE, Kedves AT, Duman RS, Taylor JR(2008) Regionally specific regulation of ERK MAP kinase in a model ofantidepressant-sensitive chronic depression. Biol Psychiatry 63:353–359.

Ide M, Lewis DA (2010) Altered cortical CDC42 signaling pathways inschizophrenia: implications for dendritic spine deficits. Biol Psychiatry68:25–32.

Ivanov A, Pellegrino C, Rama S, Dumalska I, Salyha Y, Ben-Ari Y, Medina I(2006) Opposing role of synaptic and extrasynaptic NMDA receptors inregulation of the extracellular signal-regulated kinases (ERK) activity incultured rat hippocampal neurons. J Physiol 572:789 –798.

Johnson M, Sharma M, Jamieson C, Henderson JM, Mok MT, Bendall L,Henderson BR (2009) Regulation of beta-catenin trafficking to themembrane in living cells. Cell Signal 21:339 –348.

Kasai H, Fukuda M, Watanabe S, Hayashi-Takagi A, Noguchi J (2010)Structural dynamics of dendritic spines in memory and cognition. TrendsNeurosci 33:121–129.

Kelly A, Laroche S, Davis S (2003) Activation of mitogen-activated proteinkinase/extracellular signal-regulated kinase in hippocampal circuitry isrequired for consolidation and reconsolidation of recognition memory.J Neurosci 23:5354 –5360.

Kholmanskikh SS, Koeller HB, Wynshaw-Boris A, Gomez T, Letourneau PC,Ross ME (2006) Calcium-dependent interaction of Lis1 with IQGAP1and Cdc42 promotes neuronal motility. Nat Neurosci 9:50 –57.

Kim JJ, Fanselow MS (1992) Modality-specific retrograde amnesia of fear.Science 256:675– 677.

Kim MJ, Dunah AW, Wang YT, Sheng M (2005) Differential roles of NR2A-and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPAreceptor trafficking. Neuron 46:745–760.

Kishimoto Y, Kawahara S, Kirino Y, Kadotani H, Nakamura Y, Ikeda M,Yoshioka T (1997) Conditioned eyeblink response is impaired inmutant mice lacking NMDA receptor subunit NR2A. Neuroreport8:3717–3721.

Kojima S, Vignjevic D, Borisy GG (2004) Improved silencing vector co-expressing GFP and small hairpin RNA. Biotechniques 36:74 –79.

Komiyama NH, Watabe AM, Carlisle HJ, Porter K, Charlesworth P, Monti J,Strathdee DJ, O’Carroll CM, Martin SJ, Morris RG, O’Dell TJ, Grant SG(2002) SynGAP regulates ERK/MAPK signaling, synaptic plasticity, andlearning in the complex with postsynaptic density 95 and NMDA recep-tor. J Neurosci 22:9721–9732.

Kurella VB, Richard JM, Parke CL, Lecour LF, Jr., Bellamy HD, WorthylakeDK (2009) Crystal structure of the GTPase-activating protein-relateddomain from IQGAP1. J Biol Chem 284:14857–14865.

Lamprecht R, Dracheva S, Assoun S, LeDoux JE (2009) Fear conditioninginduces distinct patterns of gene expression in lateral amygdala. GenesBrain Behav 8:735–743.

Law AJ, Weickert CS, Hyde TM, Kleinman JE, Harrison PJ (2004) Reducedspinophilin but not microtubule-associated protein 2 expression in thehippocampal formation in schizophrenia and mood disorders: molecularevidence for a pathology of dendritic spines. Am J Psychiatry161:1848 –1855.

Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, Li XY, Aghajanian G,Duman RS (2010) mTOR-dependent synapse formation underlies therapid antidepressant effects of NMDA antagonists. Science 329:959 –964.

Li S, Wang Q, Chakladar A, Bronson RT, Bernards A (2000) Gastric hyper-plasia in mice lacking the putative Cdc42 effector IQGAP1. Mol Cell Biol20:697–701.

Li Z, McNulty DE, Marler KJ, Lim L, Hall C, Annan RS, Sacks DB (2005)IQGAP1 promotes neurite outgrowth in a phosphorylation-dependentmanner. J Biol Chem 280:13871–13878.

Lim IA, Merrill MA, Chen Y, Hell JW (2003) Disruption of the NMDAreceptor-PSD-95 interaction in hippocampal neurons with no obviousphysiological short-term effect. Neuropharmacology 45:738 –754.

Luo JH, Fu ZY, Losi G, Kim BG, Prybylowski K, Vissel B, Vicini S (2002)Functional expression of distinct NMDA channel subunits tagged withgreen fluorescent protein in hippocampal neurons in culture. Neuro-pharmacology 42:306 –318.

Muhia M, Yee BK, Feldon J, Markopoulos F, Knuesel I (2010) Disruption ofhippocampus-regulated behavioural and cognitive processes by heterozy-gous constitutive deletion of SynGAP. Eur J Neurosci 31:529 –543.

Noritake J, Watanabe T, Sato K, Wang S, Kaibuchi K (2005) IQGAP1: a keyregulator of adhesion and migration. J Cell Sci 118:2085–2092.

Owen D, Campbell LJ, Littlefield K, Evetts KA, Li Z, Sacks DB, Lowe PN, MottHR (2008) The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: inter-faces differ between the complexes. J Biol Chem 283:1692–1704.

Petralia RS, Sans N, Wang YX, Wenthold RJ (2005) Ontogeny of postsyn-aptic density proteins at glutamatergic synapses. Mol Cell Neurosci29:436 – 452.

Phillips RG, LeDoux JE (1992) Differential contribution of amygdala andhippocampus to cued and contextual fear conditioning. Behav Neurosci106:274 –285.

Radley JJ, Johnson LR, Janssen WG, Martino J, Lamprecht R, Hof PR, LeDouxJE, Morrison JH (2006) Associative Pavlovian conditioning leads to anincrease in spinophilin-immunoreactive dendritic spines in the lateralamygdala. Eur J Neurosci 24:876 – 884.

Radulovic J, Kammermeier J, Spiess J (1998) Relationship between fos pro-duction and classical fear conditioning: effects of novelty, latent inhibi-tion, and unconditioned stimulus preexposure. J Neurosci 18:7452–7461.

Rama S, Krapivinsky G, Clapham DE, Medina I (2008) The MUPP1-SynGAPalpha protein complex does not mediate activity-induced LTP.Mol Cell Neurosci 38:183–188.

Reiner O, Sapoznik S, Sapir T (2006) Lissencephaly 1 linking to multiplediseases: mental retardation, neurodegeneration, schizophrenia, malesterility, and more. Neuromolecular Med 8:547–565.

Ren JG, Li Z, Crimmins DL, Sacks DB (2005) Self-association of IQGAP1:characterization and functional sequelae. J Biol Chem 280:34548 –34557.

Ren JG, Li Z, Sacks DB (2007) IQGAP1 modulates activation of B-Raf. ProcNatl Acad Sci U S A 104:10465–10469.


Ren JG, Li Z, Sacks DB (2008) IQGAP1 integrates Ca2�/calmodulin andB-Raf signaling. J Biol Chem 283:22972–22982.

Roy M, Li Z, Sacks DB (2004) IQGAP1 binds ERK2 and modulates its ac-tivity. J Biol Chem 279:17329 –17337.

Roy M, Li Z, Sacks DB (2005) IQGAP1 is a scaffold for mitogen-activatedprotein kinase signaling. Mol Cell Biol 25:7940 –7952.

Rumbaugh G, Adams JP, Kim JH, Huganir RL (2006) SynGAP regulatessynaptic strength and mitogen-activated protein kinases in cultured neu-rons. Proc Natl Acad Sci U S A 103:4344 – 4351.

Ryu J, Futai K, Feliu M, Weinberg R, Sheng M (2008) Constitutively activeRap2 transgenic mice display fewer dendritic spines, reduced extracellularsignal-regulated kinase signaling, enhanced long-term depression, andimpaired spatial learning and fear extinction. J Neurosci 28:8178 – 8188.

Sananbenesi F, Fischer A, Schrick C, Spiess J, Radulovic J (2002) Phosphor-ylation of hippocampal Erk-1/2, Elk-1, and p90-Rsk-1 during contextualfear conditioning: interactions between Erk-1/2 and Elk-1. Mol Cell Neu-rosci 21:463– 476.

Schafe GE, Atkins CM, Swank MW, Bauer EP, Sweatt JD, LeDoux JE (2000)Activation of ERK/MAP kinase in the amygdala is required for memoryconsolidation of pavlovian fear conditioning. J Neurosci 20:8177– 8187.

Schrick C, Fischer A, Srivastava DP, Tronson NC, Penzes P, Radulovic J(2007) N-cadherin regulates cytoskeletally associated IQGAP1/ERK sig-naling and memory formation. Neuron 55:786 –798.

Schulz-Schaeffer WJ (2010) The synaptic pathology of alpha-synuclein ag-gregation in dementia with Lewy bodies, Parkinson’s disease and Parkin-son’s disease dementia. Acta Neuropathol 120:131–143.

Shalin SC, Hernandez CM, Dougherty MK, Morrison DK, Sweatt JD (2006)Kinase suppressor of Ras1 compartmentalizes hippocampal signal trans-duction and subserves synaptic plasticity and memory formation. Neuron50:765–779.

Sheng M, Kim MJ (2002) Postsynaptic signaling and plasticity mechanisms.Science 298:776 –780.

Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A, Hvalby O,Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF, Sun W, WebsterLC, Grant SG, Eilers J, Konnerth A, Li J, McNamara JO, Seeburg PH(1998) Importance of the intracellular domain of NR2 subunits forNMDA receptor function in vivo. Cell 92:279 –289.

Sweatt JD (2004) Hippocampal function in cognition. Psychopharmacol-ogy (Berl) 174:99 –110.

Tu W, Xu X, Peng L, Zhong X, Zhang W, Soundarapandian MM, Balel C,Wang M, Jia N, Lew F, Chan SL, Chen Y, Lu Y (2010) DAPK1 interac-tion with NMDA receptor NR2B subunits mediates brain damage instroke. Cell 140:222–234.

Vazquez LE, Chen HJ, Sokolova I, Knuesel I, Kennedy MB (2004) SynGAPregulates spine formation. J Neurosci 24:8862– 8872.

Wang S, Watanabe T, Noritake J, Fukata M, Yoshimura T, Itoh N, Harada T,Nakagawa M, Matsuura Y, Arimura N, Kaibuchi K (2007) IQGAP3, anovel effector of Rac1 and Cdc42, regulates neurite outgrowth. J Cell Sci120:567–577.

Weissbach L, Settleman J, Kalady MF, Snijders AJ, Murthy AE, Yan YX,Bernards A (1994) Identification of a human rasGAP-related proteincontaining calmodulin-binding motifs. J Biol Chem 269:20517–20521.

Yaka R, He DY, Phamluong K, Ron D (2003) Pituitary adenylate cyclase-activating polypeptide (PACAP(1–38)) enhances N-methyl-D-aspartatereceptor function and brain-derived neurotrophic factor expression viaRACK1. J Biol Chem 278:9630 –9638.

Yuan P, Zhou R, Wang Y, Li X, Li J, Chen G, Guitart X, Manji HK (2010)Altered levels of extracellular signal-regulated kinase signaling proteins inpostmortem frontal cortex of individuals with mood disorders andschizophrenia. J Affect Disord 124:164 –169.


cellular/molecular iqgap1regulatesnr2asignaling,spinedensity, … · 2011. 6. 8. ·...

Documents